Dual regulation of metalloproteinase expression in chondrocytes by Wnt-1-inducible signaling pathway protein 3/CCN6

Objective Wnt-1inducible signaling pathway protein 3 (WISP-3)/CCN6 is mutated in progressive pseudorheumatoid dysplasia and may have effects on cartilage homeostasis. The aim of this study was to ascertain additional roles for WISP-3/CCN6 by determining its expression in osteoarthritic (OA) cartilage and by investigating its effects on cartilage-relevant metalloproteinase expression in immortalized (C-28/I2) and primary chondrocytes. Methods Cartilage steady-state levels of WISP-3/CCN6 messenger RNA and protein production were determined by real-time quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. WISP-3/CCN6 was overexpressed in C-28/I2 cells, and the resultant clones were analyzed by quantitative RT-PCR. The stable clones were analyzed by RT-PCR for metalloproteinase expression, and the signaling pathways involved were investigated using pharmacologic inhibition. The effects of WISP-3/CCN6 on metalloproteinase expression in primary chondrocytes were investigated using a small interfering RNA approach. Results WISP-3/CCN6 was highly expressed in OA cartilage compared with undamaged cartilage, at both the RNA and protein levels. WISP-3/CCN6 overexpression in C-28/I2 cells resulted in unexpected dual regulation of metalloproteinases; expression of the potent aggrecanase ADAMTS-5 was down-regulated 9-fold, while expression of MMP-10 was up-regulated 14-fold, and these responses were accentuated in the WISP-3/CCN6 clones grown in suspension. MMP-10 up-regulation was dependent on several MAPKs, but WISP-3/CCN6mediated ADAMTS-5 repression was independent of these pathways and was partially relieved by activation of beta-catenin signaling. WISP-3/CCN6 also suppressed ADAMTS-5 expression in C-28/I2 cells treated with cytokines. In cytokine-treated primary chondrocytes, gene silencing of WISP-3/CCN6 resulted in enhanced ADAMTS-5 expression, while MMP-10 expression was suppressed. Conclusion WISP-3/CCN6 was highly expressed in end-stage OA cartilage, suggesting a role for this growth factor in cartilage homeostasis. WISP-3/CCN6induced repression of ADAMTS-5 expression and regulation of MMP-10 expression suggest complex and context-dependent roles for WISP-3/CCN6 in cartilage biology.